1. Technical Field
This invention relates to the field of immunology. In particular, the invention involves methods for presenting immunogenic substances to the body in a manner that results in enhanced recognition of the substance by the immune system. The compounds and methods of the invention consequently are useful for preparation of vaccines, methods of vaccinating and vaccination for both protection from and treatment for diseases such as bacterial and viral diseases or any disease amenable to prevention or treatment by vaccine, including cancer. Diseases previously resistant to attempts to vaccinate against them, such as HIV disease are of particular interest in uses for the invention.
2. Description of the Background Art
Long-term administration of anti-viral drugs is expensive and often involves unpleasant or even dangerous side effects. Increasing numbers of pathogenic bacteria are becoming drug-resistant. Treatment of infectious disease in the current health care environment therefore often is problematic. Moreover, prevention of disease, where possible, is far preferable to treatment. Therefore, non-toxic and effective vaccines against a variety of pathogens would be highly desirable. However, no vaccine, approved as safe and effective by the U.S. Food and Drug Administration, currently exists for a number of diseases, including HIV. Even where vaccines for a particular pathologic agent do exist, more potent, more effective and safer vaccines are needed in the art.
A vaccine that can stimulate CTL and a T-help cellular immune response can be used both as a prophylactic vaccine and also as part of a treatment for those who are infected. Livingston et al., J. Immunol. 159:1352-1383, 1997; Vitiello et al., J. Clin. Invest. 95:341-349, 1995. Stimulation of CTL is important in restricting viral replication during both the acute and chronic stages of infection; these CTL responses are critical in the immunological defense against such diseases as HIV, including resistance to infection in the first instance and long-term non-progression to AIDS in infected persons. T helper responses also are necessary for an optimal immune response to any infectious disease. A vaccine which effectively produces an increase in both CTL and T helper immune responses would be of enormous use for treatment or prophylaxis of any number of diseases, particularly viral diseases such as HCMV and HIV.
Exogenous T helper activity can be provided in trans by co-administration of the pan HLA DR-binding epitope, PADRE. The PADRE sequence is a chemically defined promiscuous T helper peptide epitope capable of binding with high affinity to a broad range of the most common HLA-DR types. Vaccines using this strategy are known to require formulation with a potent adjuvant to evoke a cytolytic response. Previously, peptide administered with DNA adjuvant has been shown to stimulate CTL in a Th-independent manner, but in some cases only after repeated doses. Cho et al., J. Immunol. 168:4907-4913, 2002; Cho et al., Nat. Biotechnol. 18:509-514, 2000.
To produce a strong CTL response, it is usually necessary to formulate a vaccine with powerful immunological adjuvants. Unfortunately, many of the known adjuvants fail to induce antigen-specific CTL, and many have associated side effects which make them unsuitable for human use. Recently, DNA adjuvants have attracted attention as safe and effective for human use with the ability to promote CTL responses. Adjuvant activity has been associated with palindromic DNA sequences that contain unmethylated CpG dinucleotides which conform to the general consensus motif of XCGY, where X is any base except C and Y is any base except G. Increasing the number of stimulatory CpG motifs in an oligodeoxynucleotide (ODN) increases its activity, while the addition of a CpG on an end or in an unfavorable sequence context could actually reduce the degree of immune activation. Krieg et al., Nature 374:6546-6549, 1995. Elimination of the CpG dinucleotides from ODN abolish their stimulatory activity. When CpG is replaced with GpG, the ODN becomes inhibitory and antagonistic to the activity of the parent ODN. Hoe et al., J. Immunol. 171:4920-4926, 2003.
For activating human cells, the optimal motif is GTCGTT and the best CpG motif for mouse or rabbit immune cells is two 5′ purines followed by the CpG dinucleotide and ending with two 3′ pyrimidines. Hartmann et al., J. Immunol. 164:1617-1624, 2000. The CpG DNA directly stimulates antigen presenting cells to produce cytokines (including TNF-α, IL-1, IL-6, IL-10, IL-12 and GM-CSF) and to upregulate expression of MHC and crucial costimulatory molecules. CpG DNA also may act on B lymphocytes, inducing their proliferation and then production of IL-6 and IL-10. CpG ODN also caused enhanced cytotoxicity and enhanced IFN-γ secretion by NK cells. The effect on NK cells may be indirect, requiring the presence of adherent cells of CpG-conditioned supernatants which contain IL-12, TNF-α and Type 1 interferons.
Interspecies differences in recognition by toll-like receptors (TLR) or other immune activation recognition may result in differences in recognition of CpG motifs. For example, the mouse TLR9 molecule is preferentially activated by the CpG motif GACGTT, whereas the human TLR9 is optimally triggered by the motif GTCGTT. See Bauer et al., Proc. Natl. Acad. Sci. USA 98:9237-9242, 2001; Hartmann et al., J. Immunol. 164:1617-1624, 2000. Despite these differences, addition of a CpG ODN to a commercial hepatitis B vaccine markedly accelerated seroconversion (protective IgG antibody levels attained in 2 weeks), showing the usefulness of these types of strategies in humans. See Krieg, Trends Immunol. 23:64-65, 2002. CpG adjuvant activity (increased rapidity of response and higher titers, resulting in protective titers to hepatitis B in 89% of the subjects by 8 weeks) has also been shown in immunocompromised HIV-infected patients. This beneficial effect in a population with reduced response can be extended to benefit other groups such as the elderly, alcoholics and cancer patients, as well as the general population. Therefore, to add extra insurance that positive immune responses seen in the well-known mouse model systems are predictive of the same results in humans, experimentation with a human-specific or a primate- and human-specific CpG ODN sequence should confirm the techniques usefulness.
Without wishing to be bound by theory, TLR9 ligands (CpG ODN) may exert their effects by specifically and strongly stimulating plasmacytoid dendritic cells, which are thought to have an important role in T-cell self-tolerance to antigens. See Krieg, Nat. Med. 9(7):831-835, 2003. Adjuvants that can activate plasmacytoid dendritic cells through the TLR9 receptor may be one key to get the most out of a vaccine antigen. See Kuwana et al., Eur. J. Immunol. 31:2547-2557, 2001; Ferguson et al., J. Immunol. 168:5589-5595, 2002.
Current strategies have not yielded vaccines that induce a strong, durable CTL response which results in protection from or prevention of many diseases, including, for example HIV, HCMV and cancer, which could be useful in public health, either to prevent occurrence of disease or to promote immune attack in affected persons. Therefore, new methods of producing vaccines are needed in the art.